WO2013163431A1 - Diagnostic et traitement de tumeur cérébrale - Google Patents

Diagnostic et traitement de tumeur cérébrale Download PDF

Info

Publication number
WO2013163431A1
WO2013163431A1 PCT/US2013/038219 US2013038219W WO2013163431A1 WO 2013163431 A1 WO2013163431 A1 WO 2013163431A1 US 2013038219 W US2013038219 W US 2013038219W WO 2013163431 A1 WO2013163431 A1 WO 2013163431A1
Authority
WO
WIPO (PCT)
Prior art keywords
mdgi
brain
tumor
brain tumor
antibody
Prior art date
Application number
PCT/US2013/038219
Other languages
English (en)
Inventor
Erkki Ruoslahti
Pirjo Laakkonen
Original Assignee
Sanford-Burnham Medical Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanford-Burnham Medical Research Institute filed Critical Sanford-Burnham Medical Research Institute
Priority to US14/396,414 priority Critical patent/US9581598B2/en
Publication of WO2013163431A1 publication Critical patent/WO2013163431A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4704—Inhibitors; Supressors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the disclosed invention is generally in the field of diagnosing brain tumor in a subject and specifically in the area of using antibodies to diagnose brain tumor/deliver therapeutic agents to brain tumors.
  • the brain consists of two types of cells: neurons and glial cells.
  • Neurons are the cells that receive and send messages within the brain and make up what is referred to as gray matter (composed of cell bodies) and white matter (composed of axons).
  • Glial cells are non-neuronal cells that provide support and protection for neurons.
  • Astrocytes are a sub-type of glial cells in the brain and spinal cord. They retain their capacity for division throughout their life span, which makes them susceptible for transformation and contribute to the prevalence of astrocyte-derived tumors.
  • Brain tumors are caused by an abnormal and uncontrolled cell growth in the brain itself or as metastatic lesions of tumors in other organs.
  • Low-grade astrocytomas a class of malignant brain tumors, acquire their blood supply by co-opting existing normal blood vessels and propagating along them without initiating angiogenesis. This leads to diffuse invasion of tumor cells over long distances in the brain without formation of real tumor masses.
  • grade III astrocytomas progress to grade IV astrocytomas they grow in size, and to cope with the increased need for nutrients and oxygen they undergo an angiogenic switch.
  • Glioblastoma multiforme are the most malignant form of astrocytomas. They become highly vascularized and tumors appear more local than the low-grade astrocytomas. GBMs also retain the ability of invasive growth.
  • tumor angiogenesis is a therapeutic strategy, which has been used to treat a variety of malignant tumors.
  • Systemic anti-angiogenic treatment of malignant brain tumors has shown to increase the number of satellite tumors in experimental animal models. There are reports according to which the treatment might even encourage tumor cells to more invasive phenotype.
  • WO 2009/136007 describes a peptide, CooP, which specifically homes to intracranial, early stage astrocytoma model that grows as islets and harbors co-opted tumor vessels in the brain.
  • the peptide can be used in targeted delivery of therapeutic substances to invasive brain cancers or metastatic brain lesions and as a diagnostic tool.
  • It is an object of the present invention is to provide a method for determining the presence or grade of brain tumor in a patient.
  • It is a further object of the present invention is to provide products, which can be used in diagnosis or treatment of brain tumors or metastatic lesions in the brain.
  • compositions and methods for determining the presence and/or grade of brain tumor in a patient such as invasive brain tumor. Also provided are methods for diagnosing and treating brain tumor or metastatic brain lesions or other tumor types.
  • MDGI mammary-derived growth inhibitor
  • FBP-3 fatty acid binding protein 3
  • H-FAPB heart type fatty acid binding protein
  • the studies described herein show that MDGI is expressed in brain tumors and in particular in lesions typical for brain tumors which cannot be removed by surgery.
  • the Examples show for the first time that MDGI expression becomes upregulated in brain tumors.
  • MDGI is expressed on the vasculature of brain tumors. In other tissues (e.g., skeletal muscle, mammary fat pad, and heart) MDGI is not normally expressed on the vasculature.
  • MDGI can be used for diagnosis and treatment of brain tumors.
  • the examples described herein show that the increase of MDGI expression increases the invasion and metastasis of tumors. Therefore, by inhibiting MDGI expression or MDGI functions, the invasion and metastatic spread of cancer can be inhibited/reduced.
  • the methods provided herein for determining the presence or grade of brain tumor include, for example, determining the level of MDGI in a sample obtained from a subject, and comparing the level of MDGI in the sample with the level of MDGI in a control sample.
  • the sample can be, for example, blood or fractions thereof, typically in plasma or serum, or tumor tissue sample obtained from a patient. A difference in the amount of MDGI in the sample relative to a control sample is indicative of the presence or grade of brain tumor.
  • the level of MDGI can be determined by using anti-MDGI antibody or other molecule recognizing MDGI. In these forms the anti- MDGI antibody or other molecule recognizing MDGI can be labeled with a detectable label.
  • the methods can include, for example, the steps of conjugating the anti-MDGI antibody to a detectable label, administering the labeled anti-MDGI antibody to the patient, and detecting the label.
  • the method includes targeting pharmaceutically active agents to brain tumor or metastatic brain lesions or other tumor types, using an anti-MDGI antibody or other molecule recognizing MDGI.
  • the methods of treating brain tumors can include inhibiting MDGI expression or function in the tumor or metastatic lesion by the aid of said antibody.
  • MDGI function can be inhibited by using a molecule that inhibits MDGI expression or function.
  • MDGI expression or function can be inhibited by using a nucleic acid molecule that inhibits MDGI gene expression.
  • the methods for targeted therapy of invasive brain cancer or metastatic brain lesions, or other tumor types can include administering to a patient in need of such therapy an effective amount of a pharmaceutical composition comprising, for example, anti-MDGI antibody or shRNA or siR A for silencing MDGI expression or function.
  • the methods of treatment disclosed herein can further include combining the targeted therapy with conventional cancer therapies selected from, for example, radiation and anti-cancer or anti-angiogenic therapies.
  • the method of targeted therapy disclosed herein can be combined with surgery.
  • a method for monitoring the efficacy of brain cancer therapy or relapse following surgical removal of cancer can include monitoring MDGI levels as described herein, whereby decreasing MDGI levels following removal of cancer are indicative of efficacy and increasing MDGI levels following removal of cancer is indicative of a relapse.
  • molecules and compositions for use in inhibiting MDGI expression or function in the brain can be anti-MDGI antibodies.
  • the molecules can be nucleic acids; for example, shRNA or siRNA which inhibit MDGI expression in the brain tumor or metastatic lesion in brain.
  • compositions comprising anti-MDGI antibody, siRNA of shRNA are provided for use in treating brain tumor or metastatic lesions in the brain or other tumor types.
  • Figures 1A-1C are bar graphs of a phage screen of two ex vivo (Ev) rounds performed by incubating the phage with cell suspension from HIFko tumors followed by three in vivo (Iv) rounds with intravenously injected phage pool into intracranial HIFko astrocytoma-bearing mice. Phage enrichment is shown as fold increase over the control phage ( Figure 1A). Individual phage from the third in vivo selection round were tested for ex vivo binding to the cell suspension derived from tumor containing brain and normal brain. Graph shows phage binding to tumor brain relative to the normal brain ( Figure IB). Specificity of the CooP phage homing to the brain tumors and histologically normal brain, liver, kidney and lung tissue compared to the control phage ( Figure 1C).
  • Figures 2A and 2B show the effect of MDGI expression on the binding of the CooP phage.
  • U20S cells were transfected with the MDGI encoding pcDNA3-9E10 plasmid.
  • Figure 2A shows CooP phage and unselected library binding to U20S cells transfected with MDGI (data is expressed as fold increase over the control phage ⁇ SEM.).
  • Figure 2B shows MDGI expression in the transfected cells confirmed by Western blot analysis using an antibody against MDGI and c-Myc.
  • Figure 3A shows CooP peptide accumulation (% ID g "1 ⁇ STD) in the tumor half and the healthy half of the brain of the same animal 0.25, 1, 2 and 24 hrs after intravenous administration of 5 MBq m In-CooP.
  • Figure 3B shows biodistribution of the m In-CooP and the U 1 ln-labeled control peptide (% ID g "1 ⁇ STD) in different tissues at two hours after intravenous administration of the peptides.
  • Figure 3C shows
  • Indium-labeled anti-MDGI antibody in the brain tumor tissue following injection of anti-MDGI antibody (goat anti-MDGI, Santa Cruz Biotechnology) was injected into the tail vein of U87MG human glioma bearing mice and allowed to circulate for indicated times. The animals were sacrificed and the radioactivity was measured in the healthy half and brain tumor containing brain half of the same animal.
  • Figure 4 is a graph showing weight gain in tumor bearing immunocompromised (Balb/c nu/nu) mice, treated with saline (PBS), free chlorambusil (Cbl), and targeted drug (chlorambusil conjugated to CooP peptide; CooP-CPP-Cbl, 5 mg/kg).
  • the graph shows the normalized weight of each group of the animals. Weight on day 0 (the day of implantation) was set to 100%.
  • Mammary-derived growth inhibitor (MDGI; also known as H-FABP and FABP- 3) is a small 15-kDa cytosolic protein that belongs to the family of fatty acid-binding proteins (FABPs). Sequencing of the cDNA of MDGI has shown an open reading frame coding for a protein of 133 amino acids (Kurtz et al, J. Cell Biol, 1 10(5): 1779-89 (1990)). The sequence data is also available from EMBL/GenBank DDJB accession number X51933 or NM 004102. Expression of the fatty acid-binding proteins is relatively tissue specific.
  • MDGI Fatty acid-binding proteins avidly bind hydrophobic ligands and mediate fatty acid metabolism (Hanhoff, et al, Mol Cell Biochem, 239:45-54 (2002)).
  • MDGI is expressed at least in 39 different tissues, including the heart, brain, lung, and breast (Therry-Mieg, et al, Genome Biol, 7(Suppl 1):S12 1-4 (2006)). With respect to pathologies, MDGI is abundant in the myocardium and is released into circulation after myocardium injury.
  • MDGI can be used as a sensitive early marker to detect the myocardial damage in diseases such as acute myocardial infarction and chronic heart failure (Tanaka et al, Clin Biochem, 24: 195-201 (1991); Setsuta, et al, Am J. Med., 113 :717-722 (2002)). Moreover, MDGI is a valuable prognostic marker, since circulating concentrations of MDGI can indicate the severity of congestive heart failure (Goto et al, Heart, 89(1 1): 1303-7 (2003)). MDGI has also been suggested as a circulating marker for the stroke (Pelsers et al. Clinical Chemistry, 50(9): 1568-75 (2004)).
  • the examples described herein show that the anti-MDGI antibody accumulates in the tumor-associated vasculature after intravenous injection, whereas blood vessels in other tissues such as muscle, heart, normal brain, kidney, liver, and lung show no detectable accumulation of the antibody.
  • the studies also show that in the tumor tissue, MDGI was accessible via the blood circulation, and could therefore act as a receptor molecule for intravenously administrated antibodies or other molecules recognizing MDGI.
  • the disclosed method can take certain forms.
  • a method for determining the presence or grade of brain tumor or metastatic brain lesions or other brain tumor types in a patient comprising: (i) determining the level of MDGI in a sample obtained from a patient; (ii) comparing the level of MDGI in the sample with the level of MDGI in a control sample; wherein a difference in the amount of MDGI in the sample relative to the control sample is indicative of the presence or grade of brain tumor or other brain tumor type.
  • a method for targeted therapy of brain cancer or metastatic brain lesions, or other brain tumor types comprising administering to a patient in need of such therapy an efficient amount of a
  • composition comprising anti-MDGI antibody or siRNA or shRNA for MDGI.
  • a method for diagnosing brain tumor or metastatic brain lesion or other brain tumor types in a patient comprising the steps of: conjugating an anti-MDGI antibody to a detectable label, administering the labeled anti- MDGI antibody to the patient, and detecting the label.
  • the use of the MDGI levels obtained in the disclosed methods for monitoring the efficacy of therapy or relapse after the surgical removal of the brain tumor or metastatic lesion or other type of tumor tissue in brain.
  • the sample is selected from the group consisting whole blood, blood plasma, serum or tumor tissue.
  • the level of MDGI is determined by using anti-MDGI antibody or other molecule recognizing MDGI.
  • the anti-MDGI antibody or other molecule is labeled with a detectable label, such as an imaging agent.
  • the method further comprises combining the said targeted therapy with cancer therapies.
  • compositions can also take certain forms.
  • anti-MDGI antibody for use in diagnosing or treating brain tumor or metastatic brain lesions or other brain tumor types.
  • anti-MDGI antibody for use in targeting pharmaceutically active agents to brain tumor or metastatic brain lesions or other brain tumor types.
  • anti-MDGI antibody for use in inhibiting MDGI expression or function in the tumor or metastatic lesion by the aid of said antibody.
  • siRNA or shRNA for inhibiting MDGI expression or function in the tumor or metastatic lesion in a brain, metastatic brain lesions or other brain tumor types.
  • a composition comprising anti-MDGI antibody or siRNA or shRNA for silencing MDGI for use in treating brain tumor or metastatic lesions in the brain or other brain tumor types.
  • Also disclosed are methods comprising administering a labeled anti-MDGI antibody to a patient, where the labeled anti-MDGI antibody comprises an anti-MDGI antibody conjugated to a detectable label, and detecting the label.
  • the presence and/or location of the label indicates the presence and/or location of brain tumor or metastatic brain lesions or other brain tumor types in the patient.
  • the method can further comprise determining the level of MDGI in a sample obtained from a patient by quantitating the detected label, and comparing the level of MDGI in the sample with the level of MDGI in a control sample. A difference in the amount of MDGI in the sample relative to the control sample is indicative of the presence or grade of brain tumor or metastatic brain lesions or other brain tumor type.
  • the label can be detected via imaging of the brain.
  • the method can further comprise surgical removal of the brain tumor or metastatic brain lesions or other brain tumor types guided by the location of the label.
  • the method can further comprise administering to the patient an effective amount of a pharmaceutical composition comprising anti-MDGI antibody or siRNA or shRNA for MDGI.
  • the method can further comprise combining the administration of the pharmaceutical composition with cancer therapies.
  • the method can further comprise monitoring the efficacy of therapy or relapse after the surgical removal of the brain tumor or metastatic brain lesion or other type of tumor tissue in brain.
  • the efficacy of therapy or relapse can be monitored by detecting the presence and/or location of brain tumor or metastatic brain lesions or other brain tumor types in the patient by the presence and/or location of a labeled anti-MDGI antibody administered to the patient.
  • Also disclosed are methods comprising administering to a patient in need of such therapy an effective amount of a pharmaceutical composition comprising anti-MDGI antibody or siRNA or shRNA for MDGI.
  • the method can further comprise combining the administration of the pharmaceutical composition with cancer therapies.
  • brain tissue of the patient can exhibit detectable MDGI.
  • Also disclosed are methods comprising determining the level of MDGI in a sample obtained from a patient, comparing the level of MDGI in the sample with the level of MDGI in a control sample, and administering an effective amount of a pharmaceutical composition comprising anti-MDGI antibody or siRNA or shRNA for MDGI in patients in which a difference in the amount of MDGI in the sample relative to the control sample is detected.
  • the method can further comprise combining the administration of the pharmaceutical composition with cancer therapies.
  • CooP peptides for use in diagnosing or treating brain tumor or metastatic brain lesions or other brain tumor types. Also disclosed are CooP peptides for use in targeting pharmaceutically active agents to brain tumor or metastatic brain lesions or other brain tumor types. Also disclosed are CooP peptides for use in inhibiting MDGI expression or function in a brain tumor or metastatic brain lesions or other brain tumor types by the aid of said antibody. Also disclosed is use of the MDGI levels obtained in the disclosed method for monitoring the efficacy of therapy or relapse after the surgical removal of the brain tumor or metastatic brain lesion or other type of tumor tissue in brain.
  • MDGI presence, location, and/or levels can be detected.
  • the detected MDGI presence, location, and/or levels can be used for a variety of purposes.
  • the presence and/or levels of MDGI can indicate the presence or grade of brain tumors or metastatic brain lesions or other brain tumor types.
  • the location of MDGI can indicate where cancer cells or tumors are present; in particular, locations where more severe or dangerous (e.g., metastatic) cancer cells or tumors are present.
  • the presence, location, and/or levels of MDGI can indicate, for example, whether the patient's cancer is better or worse and/or is or is not responding to therapy.
  • detection of the presence, location, and/or levels of MDGI can be used to monitor therapy and/or the condition of patient's disease. In this way, the patient's prognosis can be determined, estimated, and/or assessed.
  • detection of the presence, location, and/or levels of MDGI can be accomplished using compounds or compositions that bind to, target, and/or home to MDGI.
  • anti-MDGI antibodies and/or peptides that bind to MDGI such as CooP peptide
  • a label in the conjugate can then be used to detect, quantitate, and/or image MDGI present in tissue and/or samples.
  • Some forms of the disclosed methods and compositions can be used to treat brain tumors, metastatic brain lesions, and/or other brain tumor types.
  • the location of brain tumors, metastatic brain lesions, and/or other brain tumor types determined using the disclosed methods can be used to guide surgical removal of the brain tumors, metastatic brain lesions, and/or other brain tumor types.
  • administered therapeutics can be targeted to brain tumors, metastatic brain lesions, and/or other brain tumor types using compounds or compositions that bind to, target, and/or home to MDGI.
  • anti-MDGI antibodies and/or peptides that bind to MDGI such as CooP peptide
  • a therapeutic compound in the conjugate can then have an effect on the brain tumor, metastatic brain lesions, and/or other brain tumor type.
  • Activity refers to a biological activity.
  • conjugate refers to a compound comprising two or more molecules bound together, optionally through a linking group, to form a single structure.
  • the binding can be made by a direct connection (e.g., a chemical bond) between the molecules or by use of a linking group.
  • Effective amount of a compound as used herein is refers to a nontoxic but sufficient amount of the compound to provide the desired result. As will be pointed out elsewhere herein, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.
  • an efficient amount of a pharmaceutical composition is meant an amount efficient to have an effect to the condition of the patient.
  • the dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician based on the clinical condition of the subject involved.
  • the dose, schedule of doses and route of administration can be varied.
  • the efficacy of administration of a particular dose of the compounds or compositions according to the methods described herein can be determined by evaluating the particular aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field.
  • a subject's physical condition is shown to be improved (e.g., a tumor has partially or fully regressed)
  • the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.
  • a “grade of brain tumor” as used herein refers to the classification of brain tumors, which is based on the premise that each type of tumor results from the abnormal growth of a specific cell type. To the extent that the behavior of a tumor correlates with basic cell type, tumor classification dictates the choice of therapy and predicts prognosis. For example, WHO grading of astrocytic tumors divide the tumors to 4 grades:
  • “Inhibit” as used herein means to reduce or decrease in activity or expression. This can be a complete inhibition of activity or expression, or a partial inhibition.
  • Inhibition can be compared to a control or to a standard level.
  • Inhibition can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the control or standard level.
  • In need of treatment refers to a judgment made by a caregiver (e.g., physician, nurse, nurse practitioner, or individual in the case of humans;
  • Non-natural amino acid refers to an organic compound that has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid.
  • the non-natural amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., selectivity, stability) when the non-natural amino acid is either substituted for a natural amino acid or incorporated into a peptide.
  • Modulate refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control.
  • activities can increase or decrease as compared to controls in the absence of these compounds.
  • an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
  • a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
  • a compound that increases a known activity is an "agonist”.
  • One that decreases, or prevents, a known activity is an "antagonist.”
  • Monitoring refers to any method in the art by which an activity can be measured.
  • Patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • Peptide refers to a class of compounds composed of amino acids chemically bound together. In general, the amino acids are chemically bound together via amide linkages (CONH); however, the amino acids may be bound together by other chemical bonds known in the art. For example, the amino acids may be bound by amine linkages. Peptide as used herein includes oligomers of amino acids and small and large peptides, including polypeptides.
  • “Pharmaceutically acceptable” is used herein to refer to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • “Pharmacological activity” as used herein refers to the inherent physical properties of a peptide or polypeptide. These properties include but are not limited to half-life, solubility, and stability and other pharmacokinetic properties.
  • Providing refers to any means or manner of adding a compound or molecule to something else, such as something known in the art. Examples of providing can include the use of pipettes, pipettemen, syringes, needles, tubing, guns, etc. This can be manual or automated. It can include transfection by any means or manner of providing nucleic acids to, for example, dishes, cells, tissue, cell-free systems and can be in vitro or in vivo.
  • Preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
  • sample refers to a biological sample from a patient, preferably a blood or fractions of blood sample, typically a plasma or serum or tumor tissue sample obtained from a patient diagnosed, suspected, or postulated to have brain tumor, metastatic brain lesions, or other brain tumor types.
  • a control sample is meant a biological sample from a person not having the disease the patient is diagnosed or postulated to have.
  • siRNA refers to a small interfering RNA, commonly 18 to 30 nucleotides, preferably 20 to 25, more preferably 21 to 23, or approximately 22 nucleotide double- stranded RNA. Preferably at least one strand has a 5 ' - and/or 3 Overhang of 1 to 5, preferably 1 to 3, or 2 nucleotides. siRNA is involved in the RNA interference pathway where the siRNA interferes with the expression of a specific gene.
  • shRNA refers to a short hairpin RNA structure that forms a tight hairpin turn, which can also be used to silence gene expression via RNA interference.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA, which matches the siRNA that is bound to it.
  • RISC RNA-induced silencing complex
  • RNA-mediated inhibition of via RNA interference refers to RNA-mediated inhibition of via RNA interference. Silencing generally is mediated by the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • Subject as used herein includes, but is not limited to a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, etc.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • Treatment and “treating” are meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • treatment while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention.
  • the effects of treatment can be measured or assessed as described herein and as known in the art
  • compositions containing an anti-MDGI antibody, or a peptide or other molecule recognizing MDGI, and a detectable label are provided.
  • the composition can be used in diagnosing invasive brain tumor or metastatic brain lesions or other brain tumor types.
  • compositions described herein are also useful for targeted therapy of invasive brain cancer or metastatic brain lesions, or other tumor types comprising administration of an effective amount of a pharmaceutical composition to a patient.
  • compositions described herein are also useful for targeted therapy of invasive brain cancer or metastatic brain lesions, or other tumor types comprising administration of an efficient amount of a pharmaceutical composition to a patient.
  • anti-MDGI antibody and nucleic acids disclosed herein are conjugated to CooP.
  • the anti-MDGI antibodies described herein can also be used for targeted delivery of cancer therapeutics to the brain.
  • the antibodies, peptide or other molecule can be provided as a pharmaceutical composition, in association with a pharmaceutically active agent.
  • a pharmaceutical composition can be prepared by combining a pharmaceutically acceptable excipient, carrier, diluents, buffer, stabilizer or other materials well known to those skilled in the art with the pharmaceutically active agent, e.g., anti-MDGI antibody, other molecule binding to MDGI.
  • the composition is suitable to be administrated via a suitable route, preferably a solution for injection intravenously to a patient.
  • anti-MDGI antibody or other molecules recognizing MDGI or a pharmaceutical composition comprising them may be administrated via various routes, typically via intravenous or oral administration or tissue injection. Preferably they are administrated via intravenous administration, (i) Anti-MDGI Antibodies and other molecules binding to MDGI
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.
  • concentration of the anti-MDGI antibody or other molecule in a pharmaceutical composition may vary in wide ranges and depends for example on the tumor type, grade of tumor, nature of the antibody, other molecule or peptide or condition of the patient. Generally the administrated amount is 0.01 - 100 mg/kg, typically 0.1 - 50 mg/kg.
  • CooP is a nine amino acid long peptide CGLSGLGVA (SEQ ID NO:5).
  • Antibodies recognizing MDGI are commercially available, for example from
  • an antibody used in the disclosed methods is MDGI in a biological sample is a monoclonal antibody.
  • a monoclonal antibody composition is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) only one type of antibody molecule.
  • the hybridoma cell is formed by fusing an antibody- producing cell and a myeloma or other self-perpetuating cell line.
  • Such antibodies were first described by Kohler and Milstein, Nature, 256:495-497 (1975), the disclosure of which is herein incorporated by reference.
  • An exemplary hybridoma technology is described by Niman et al, Proc. Natl. Acad. Sci. U.S.A., 80:4949-4953 (1983).
  • Monoclonal antibodies specific for MDGI can be used in screening assays to detect the presence of this molecule in both liver tumor tissues and serum of
  • HCC hepatocellular carcinoma
  • monoclonal antibodies can also be used as vehicles to deliver cargos, such as nucleic acid, drug or toxin, to liver tumor cells with high expression of CDH17 on cell surface or simply as MDGI antagonists to inhibit MDGI activity in the tumor cell.
  • cargos such as nucleic acid, drug or toxin
  • hybridoma cell lines provide unlimited source for producing monoclonal antibodies when needed. Culturing the hybridoma cells can produce large quantities of the antibodies economically.
  • the antibodies or peptides recognizing MDGI may be immobilized on a carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, well, beads, disc, sphere, etc.
  • the immobilized antibody may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • Variants of the CooP peptide can also be used.
  • isolated peptides comprising an amino acid segment comprising the amino acid sequence of SEQ ID NO: 5.
  • the isolated peptides can comprise, for example, an amino acid segment comprising, for example, the amino acid sequence of SEQ ID NO: 5 having one or more conservative amino acid substitutions.
  • the amino acid segment can comprise an amino acid sequence at least about 90%, 80%, 70%, or 60% identical to the amino acid sequence of SEQ ID NO: 5 or any percentage in between that represents a change, including addition or deletion, of one or more amino acid.
  • the amino acid segment can comprise the amino acid sequence of SEQ ID NO: 5.
  • the amino acid segment can comprise the amino acid sequence of SEQ ID NO: 5 having one, two, three, four, five, six, seven, eight, or nine conservative amino acid substitutions.
  • the disclosed peptides can consist of the amino acid segment.
  • the amino acid segment can be, for example, non-circular, linear, circular or cyclic.
  • the amino acid segment can be circularized or cyclized via any suitable linkage, for example, a disulfide bond.
  • the peptide can have any suitable length, such as a length of less than 100 residues.
  • the peptide can have a length of, for example, less than 50 residues.
  • the peptide can have a length of, for example, less than 20 residues.
  • the disclosed peptides can selectively home to tissue expressing MDGI, such as brain tumors and metastatic brain lesions.
  • tissue expressing MDGI such as brain tumors and metastatic brain lesions.
  • the disclosed peptides can selectively interact with such tissue or tumors.
  • isolated peptides which have a length of less than 100 residues and which include the amino acid sequence SEQ ID NO: 5 or a peptidomimetic thereof.
  • Such an isolated peptide can have, for example, a length of less than 50 residues or a length of less than 20 residues.
  • disclosed can be a peptide that includes the amino acid sequence SEQ ID NO: 5 and has a length of less than 20, 50 or 100 residues.
  • the disclosed peptides can be in isolated form.
  • isolated means a peptide that is in a form that is relatively free from material such as contaminating polypeptides, lipids, nucleic acids and other cellular material that normally is associated with the peptide in a cell or that is associated with the peptide in a library or in a crude preparation.
  • the disclosed peptides can have any suitable length.
  • the disclosed peptides can have, for example, a relatively short length of less than ten, 11, 12, 13, 14, 15, 20, 25, 30, 35 or 40 residues.
  • the disclosed peptides also can be useful in the context of a significantly longer sequence.
  • the peptides can have, for example, a length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues.
  • a peptide can have a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues.
  • a peptide can have a length of 5 to 200 residues, 5 to 100 residues, 5 to 90 residues, 5 to 80 residues, 5 to 70 residues, 5 to 60 residues, 5 to 50 residues, 5 to 40 residues, 5 to 30 residues, 5 to 20 residues, 5 to 15 residues, 5 to 10 residues, 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30 residues.
  • the term "residue” refers to an amino acid or amino acid analog.
  • a “conservative variant” is a sequence in which a first amino acid is replaced by another amino acid or amino acid analog having at least one biochemical property similar to that of the first amino acid; similar properties include, for example, similar size, charge, hydrophobicity or hydrogen-bonding capacity.
  • a conservative variant can be a sequence in which a first uncharged polar amino acid is conservatively substituted with a second (non-identical) uncharged polar amino acid such as cysteine, serine, threonine, tyrosine, glycine, glutamine or asparagine or an analog thereof.
  • a conservative variant also can be a sequence in which a first basic amino acid is conservatively substituted with a second basic amino acid such as arginine, lysine, histidine, 5-hydroxylysine, N-methyllysine or an analog thereof.
  • a conservative variant can be a sequence in which a first hydrophobic amino acid is conservatively substituted with a second hydrophobic amino acid such as alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine or tryptophan or an analog thereof.
  • a conservative variant can be a sequence in which a first acidic amino acid is conservatively substituted with a second acidic amino acid such as aspartic acid or glutamic acid or an analog thereof; a sequence in which an aromatic amino acid such as phenylalanine is conservatively substituted with a second aromatic amino acid or amino acid analog, for example, tyrosine; or a sequence in which a first relatively small amino acid such as alanine is substituted with a second relatively small amino acid or amino acid analog such as glycine or valine or an analog thereof.
  • the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution.
  • a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as, for example, Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
  • this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e.
  • nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences.
  • degenerate nucleic acids encoding the disclosed variants and derivatives of the protein sequences.
  • chimeric proteins containing a disclosed peptide fused to a heterologous protein.
  • the heterologous protein can have a therapeutic activity such as cytokine activity, cytotoxic activity or pro-apoptotic activity.
  • the heterologous protein can be an antibody or antigen-binding fragment thereof.
  • the chimeric protein includes a peptide containing the amino acid sequence SEQ ID NO: 5, or a conservative variant or peptidomimetic thereof, fused to a heterologous protein.
  • heterologous as used herein in reference to a protein fused to the disclosed peptides, means a protein derived from a source other than the gene encoding the peptide or from which the peptidomimetic is derived.
  • the disclosed chimeric proteins can have a variety of lengths including, but not limited to, a length of less than 100 residues, less than 200 residues, less than 300 residues, less than 400 residues, less than 500 residues, less than 800 residues or less than 1000 residues.
  • chimera and “chimeric” refer to any combination of sequences derived from two or more sources. This includes, for example, from single moiety of subunit (e.g., nucleotide, amino acid) up to entire source sequences added, inserted and/or substituted into other sequences. Chimeras can be, for example, additive, where one or more portions of one sequence are added to one or more portions of one or more other sequences; substitutional, where one or more portions of one sequence are substituted for one or more portions of one or more other sequences; or a combination.
  • subunit e.g., nucleotide, amino acid
  • Constant substitutional chimeras can be used to refer to substitutional chimeras where the source sequences for the chimera have some structural and/or functional relationship and where portions of sequences having similar or analogous structure and/or function are substituted for each other. Typical chimeric and humanized antibodies are examples of conservative substitutional chimeras.
  • bifunctional peptides which contains a homing peptide fused to a second peptide having a separate function.
  • Such bifunctional peptides have at least two functions conferred by different portions of the full-length molecule and can, for example, display anti-cancer activity or pro-apoptotic activity in addition to selective homing activity.
  • isolated multivalent peptides that include at least two subsequences each independently containing a homing molecule (for example, the amino acid sequence SEQ ID NO: 5, or a conservative variant or peptidomimetic thereof).
  • the multivalent peptide can have, for example, at least three, at least five or at least ten of such subsequences each independently containing a homing molecule (for example, the amino acid sequence of SEQ ID NO: 5, or a conservative variant or peptidomimetic thereof).
  • the multivalent peptide can have two, three, four, five, six, seven, eight, nine, ten, fifteen or twenty identical or non-identical
  • the multivalent peptide can contain identical subsequences, which consist of a homing molecule (for example, the amino acid sequence SEQ ID NO: 5, or a conservative variant or peptidomimetic thereof).
  • the multivalent peptide contains contiguous identical or non- identical subsequences, which are not separated by any intervening amino acids.
  • the multivalent peptide can be cyclic or otherwise conformationally constrained. In one example, the peptide can be circularized or cyclized via a disulfide bond.
  • an isolated peptide, or a homing molecule as discussed further elsewhere herein can be cyclic or otherwise conformationally constrained.
  • a "conformationally constrained" molecule such as a peptide, is one in which the three-dimensional structure is maintained substantially in one spatial arrangement over time. Conformationally constrained molecules can have improved properties such as increased affinity, metabolic stability, membrane permeability or solubility. Methods of conformational constraint are well known in the art and include cyclization as discussed further elsewhere herein.
  • cyclic means a structure including an intramolecular bond between two non-adjacent amino acids or amino acid analogues.
  • the cyclization can be effected through a covalent or non-covalent bond.
  • Intramolecular bonds include, but are not limited to, backbone to backbone, side-chain to backbone and side-chain to side-chain bonds.
  • a preferred method of cyclization is through formation of a disulfide bond between the side-chains of non-adjacent amino acids or amino acid analogs.
  • Residues capable of forming a disulfide bond include, for example, cysteine (Cys), penicillamine (Pen), ⁇ , ⁇ -pentamethylene cysteine (Pmc), ⁇ , ⁇ - pentamethylene ⁇ -mercaptopropionic acid (Pmp) and functional equivalents thereof.
  • a peptide also can cyclize, for example, via a lactam bond, which can utilize a side-chain group of one amino acid or analog thereof to form a covalent attachment to the N-terminal amine of the amino-terminal residue.
  • Residues capable of forming a lactam bond include aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), ornithine (orn), ⁇ , ⁇ -diamino-propionic acid, ⁇ -amino-adipic acid (Adp) and M- (aminomethyl)benzoic acid (Mamb).
  • Cyclization additionally can be effected, for example, through the formation of a lysinonorleucine bond between lysine (Lys) and leucine (Leu) residues or a dityrosine bond between two tyrosine (Tyr) residues.
  • Lys lysine
  • Leu leucine
  • Tyr tyrosine residues
  • compositions comprising anti-MDGI antibody or siRNA or shRNA for silencing MDGI for use in treating other tumor types.
  • an antisense nucleic acid sequence can include a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to the CDH17 mRNA.
  • the antisense nucleic acid can be complementary to an entire coding strand of a target sequence, or to only a portion thereof.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence within the MDGI mRNA.
  • An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • MDGI expression can be inhibited using ribozymes.
  • a ribozyme having specificity for MDGI mRNA can include one or more sequences complementary to a nucleotide sequence within the MDGI mRNA, and a sequence having a known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff, et al., Nature 334:585-591 (1988)).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the mRNA encoded by a uORF of an extended, overlapping 5'-UTR AS mRNA species (see, e.g., U.S. Pat. No. 4,987,071 and No. 5, 116,742).
  • MDGI mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel, et al, Science, 261 : 141 1-1418 (1993)).
  • compositions described herein can be combined with conventional cancer therapeutics.
  • Such therapies are, for example, radiation and anti-cancer or anti- angiogenic therapies.
  • the method further comprises that the targeted therapy is combined with surgery.
  • a "cancer chemotherapeutic agent” is a chemical agent that inhibits the proliferation, growth, life-span or metastatic activity of cancer cells.
  • Such a cancer chemotherapeutic agent can be, without limitation, a taxane such as docetaxel; an anthracyclin such as doxorubicin; an alkylating agent; a vinca alkaloid; an anti-metabolite; a platinum agent such as cisplatin or carboplatin; a steroid such as methotrexate; an antibiotic such as adriamycin; a isofamide; or a selective estrogen receptor modulator; an antibody such as trastuzumab.
  • a taxane such as docetaxel
  • an anthracyclin such as doxorubicin
  • an alkylating agent such as doxorubicin
  • a vinca alkaloid an anti-metabolite
  • a platinum agent such as cisplatin or carboplatin
  • a steroid such as methotrexate
  • an antibiotic such as adriamycin
  • a isofamide or a selective estrogen receptor modul
  • An alkylating agent such as melphalan or chlorambucil also can be a cancer chemotherapeutic agent useful in a conjugate.
  • a vinca alkaloid such as vindesine, vinblastine or vinorelbine; or an antimetabolite such as 5-fluorouracil, 5- fluorouridine or a derivative thereof can be a cancer chemotherapeutic agent useful in a conjugate.
  • a platinum agent also can be a cancer chemotherapeutic agent useful in the compositions or conjugates described herein.
  • a platinum agent can be, for example, cisplatin or carboplatin as described, for example, in Crown, Seminars in Oncol. 28:28- 37 (2001).
  • Other cancer chemotherapeutic agents useful in a conjugate include, without limitation, methotrexate, mitomycin-C, adriamycin, ifosfamide and ansamycins.
  • Useful cytotoxic agents include, without limitation, small molecules, polypeptides, peptides, peptidomimetics, nucleic acid-molecules, cells and viruses.
  • useful cytotoxic agents include cytotoxic small molecules such as doxorubicin, docetaxel or trastuzumab; antimicrobial peptides such as those described further below; pro- apoptotic polypeptides such as caspases and toxins, for example, caspase-8; diphtheria toxin A chain, Pseudomonas exotoxin A, cholera toxin, ligand fusion toxins such as DAB389EGF, ricinus communis toxin (ricin); and cytotoxic cells such as cytotoxic T cells. See, for example, Martin et al, Cancer Res.
  • conjugates which include a moiety and a homing molecule, such as CooP.
  • CooP is used to target delivery of the moiety to the brain.
  • the term "moiety" is used broadly to mean a physical, chemical, or biological material that generally imparts a biologically useful function to a linked molecule.
  • the disclosed are conjugates can contain a therapeutic agent linked to a homing molecule that selectively homes to brain tumor.
  • the moiety is a molecule which inhibits the expression or function of MDGI as described herein, that is usefully targeted to the target of the homing molecule (e.g., CooP).
  • the conjugates described herein include anti- MDGI antibodies and/or the inhibitory nucleic acids described herein, which inhibit the expression or function of MDGI.
  • homing molecule as used herein, means any molecule that selectively homes in vivo to a target or site of interest in preference to normal tissue.
  • homoing peptide means a peptide that selectively homes in vivo to a target or site of interest. It is understood that a homing molecule that selectively homes in vivo to a target or site of interest can exhibit preferential homing to such a target or site.
  • selective homes is meant that, in vivo, the homing molecule binds preferentially to the target as compared to non-target.
  • a homing molecule can selectively home, for example, to a site where MDGI is preferentially expressed and/or accessibly expressed.
  • Selective homing to, for example, a target or site of interest generally is characterized by at least a two-fold greater localization at targets or sites of interest, as compared to several tissue types of tissue not a target or site of interest.
  • a homing molecule can be characterized by 5-fold, 10-fold, 20-fold or more preferential localization to a target or site of interest as compared to several or many tissue types that are not the target or site of interest, or as compared to-most or all non-target tissue.
  • a homing molecule homes, in part, to one or more normal organs in addition to homing to the targeted molecule, site, or tissue.
  • Selective homing can also be referred to as targeting.
  • a variety of therapeutic agents can also be included in the conjugates including, without limitation, cancer chemotherapeutic agents, cytotoxic agents, anti-angiogenic agents, polypeptides, nucleic acid molecules and small molecules.
  • a conjugate containing multiple homing molecules can include, for example, two or more, three or more, five or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, or 1000 or more homing molecules.
  • Moieties useful in a conjugate incorporating multiple homing molecules include, without limitation, phage, retroviruses, adenoviruses, adeno- associated viruses and other viruses, liposomes.
  • Other moieties include polymeric matrices, non-polymeric matrices, particles such as gold particles, microdevices, nanodevices, and nano-scale semiconductor materials.
  • a conjugate can contain, for example, a liposome or other polymeric matrix linked to at least two homing molecules. If desired, the liposome or other polymeric matrix can be linked to at least ten, at least 100 or at least 1000 homing molecules.
  • Liposomes can be useful in such conjugates; liposomes consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla. (1984)).
  • the liposome or other polymeric matrix can optionally include another component such as, without limitation, a therapeutic agent, cancer chemotherapeutic agent, cytotoxic agent, anti-angiogenic agent, polypeptide or nucleic acid molecule.
  • a therapeutic agent such as, without limitation, a therapeutic agent, cancer chemotherapeutic agent, cytotoxic agent, anti-angiogenic agent, polypeptide or nucleic acid molecule.
  • compositions can be combined, linked and/or coupled in any suitable manner.
  • moieties and homing molecules can be associated covalently or non-covalently, directly or indirectly, with or without a linker moiety,
  • MDGI inhibitors can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise a pharmaceutically effective amount of a MDGI - inhibiting nucleic acid molecule, peptide, or antibody and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the compounds described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds described herein and which is incorporated by reference herein.
  • humans and non-humans including solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the polynucleotide MDGI inhibitors are prepared with carriers that will protect against rapid elimination from, or degradation in, the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques.
  • Liposomal suspensions (including liposomes targeted to antigen-presenting cells with monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • the polynucleotide MDGI inhibitors can be inserted into genetic constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art.
  • the pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the polynucleotide delivery system.
  • MDGI expression or function is inhibited in the tumor or metastatic brain lesion by the aid of an anti-MDGI antibody.
  • the anti-MDGI antibody, peptides or other molecules recognizing MDGI can be labeled with a detectable label.
  • a detectable label is for example a fluorescence label or radiolabel.
  • single photon emission computed tomography-computed tomography (SPECT/CT) imaging is used to visualize brain tumors, when the antibody or a peptide or other molecule recognizing MDGI is labeled with m Indium.
  • SPECT/CT single photon emission computed tomography-computed tomography
  • a host mammal is inoculated with a MDGI protein or peptide and then boosted. Spleens are collected from inoculated mammals a few days after the final boost. Cell suspensions from the spleens are fused with a tumor cell in accordance with the general method described by Kohler and Milstein (Nature, 1975, 256:495-497).
  • a peptide fragment must contain sufficient amino acid residues to define the epitope of the MDGI molecule being detected. If the fragment is too short to be immunogenic, it may be conjugated to a carrier molecule.
  • suitable carrier molecules include keyhole limpet hemocyanin and bovine serum albumin.
  • Conjugation may be carried out by methods known in the art.
  • One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.
  • the peptide fragments may be synthesized by methods known in the art. Some suitable methods are described by Stuart and Young in “Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical Company (1984).
  • isolated native MDGI or recombinant MDGI may be utilized to prepare antibodies, monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain F v molecule (Ladne et al, U.S. Patent No.
  • antibodies used in the disclosed methods are reactive against MDGI if they bind with a K a of greater than or equal to 10 7 M.
  • K a of greater than or equal to 10 7 M.
  • sandwich immunoassay mouse polyclonal antibodies and rabbit polyclonal antibodies are utilized.
  • Preferred binding epitopes may be identified from a known MDGI gene sequence and its encoded amino acid sequence and used to generate MDGI antibodies with high binding affinity. Also, identification of binding epitopes on MDGI can be used in the design and construction of preferred antibodies. For example, a DNA encoding a preferred epitope on MDGI may be recombinantly expressed and used to select an antibody which binds selectively to that epitope. The selected antibodies then are exposed to the sample under conditions sufficient to allow specific binding of the antibody to the specific binding epitope on MDGI and the amount of complex formed then detected. Specific antibody methodologies are well understood and described in the literature. A more detailed description of their preparation can be found, for example, in Practical Immunology, Butt, W. R., ed., Marcel Dekker, New York, 1984.
  • Purification of the antibodies or fragments can be accomplished by a variety of methods known to those of skill including, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immunoaffinity chromatography as well as gel filtration, zone electrophoresis, etc.
  • An antibody specific for MDGI can be labeled with a detectable substance and localized in biological samples based upon the presence of the detectable substance. Examples of detectable substances include, but are not limited to,
  • radioisotopes e.g., H, C, . S, I, I, Indium
  • fluorescent labels e.g., FITC rhodamine, lanthanide phosphors
  • luminescent labels such as luminol
  • enzymatic labels e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkalline phosphatase, acetylcholinestease
  • biotinyl groups which can be detected by marked avidin, e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods
  • predetermined polypeptide epitopes recognized by a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against MDGI.
  • the molecule inhibiting MDGI expression or function can be a nucleic acid molecule. MDGI expression or function can be inhibited in the tumor or metastatic brain lesion e.g. by the aid of RNAi technology.
  • RNAi R A interference
  • RNAi R A interference
  • RNAi is a powerful approach for reducing expression of endogenously expressed proteins. It is widely used for biological applications and is being harnessed to silence mRNAs encoding pathogenic proteins for therapy.
  • RNAi is a conserved mechanism of post-transcriptional gene silencing in which small, double-stranded RNAs (siRNA or shRNA) suppress expression of genes bearing a partially complementary sequence. siRNAs can be designed to knock down any known gene.
  • siRNA and shRNA are preferably chemically synthesized.
  • shRNA or siRNA products are commercially available from several suppliers, for example from Santa Cruz Biotechnology, Inc.
  • MDGI expression can also be inhibited using an antisense nucleic acid.
  • Antisense nucleic acid sequences and delivery methods are well known in the art (Goodchild, Curr. Opin. Mol. Ther., 6(2): 120-128 (2004); Clawson, et al., Gene Ther., 1 1(17): 1331-1341 (2004)), which are incorporated herein by reference in their entirety.
  • An antisense nucleic acid sequence can be designed such that it is complementary to the entire MDGI mRNA sequence, but can also be an oligonucleotide that is antisense to only a portion of the CDH17 mRNA.
  • the antisense oligonucleotide can be complementary to a portion of the MDGI enzymatic domain (inositol 5'-phosphatase domain) or a portion of the amino-terminal src -homology domain (SH2).
  • An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense
  • oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • antisense oligonucleotides include an alpha-anomeric nucleic acid.
  • An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta- units, the strands run parallel to each other (Gaultier et al, Nucleic Acids. Res., 15:6625- 6641 (1987)).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. Nucleic Acids Res., 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al. FEBS Lett., 215:327-330 (1987)).
  • Ribozymes can also be employed to inhibit MDGI expression. Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. Ribozymes and methods for their delivery are well known in the art (Hendry, et al, BMC Chem. Biol, 4(1): 1 (2004); Grassi, et al, Curr. Pharm. Biotechnol, 5(4):369-386 (2004); Bagheri, et al, Curr. Mol. Med, 4(5):489-506 (2004); Kashani-Sabet M., Expert Opin. Biol.
  • Ribozymes By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme genes can be introduced into cells through gene-delivery mechanisms known in the art.
  • compositions described herein can be administered using several routes of administration.
  • the compounds and pharmaceutical compositions described herein can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation) and transmucosal.
  • Parenteral administration, if used, is generally
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Another approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • Vectors for targeting molecules across the blood brain barrier are known in the art.
  • lentiviral vectors including a gene of interest
  • the MDGI inhibitory molecules described herein are preferably targeted to brain tumors by conjugation to CooP.
  • Nanoparticles, liposomes and other cationic lipid molecules can also be used to deliver siRNA into animals. It has been shown that siRNAs delivered systemically in a liposomal formulation can silence the disease target apolipoprotein B (ApoB) in non- human primates (Zimmermann, et al, Nature, 2006, 441 : 11 1-1 14). A gel-based agarose/liposome/siRNA formulation is also available (Jiamg, et al., Oligonucleotides,
  • nanoparticles, liposomes, other cationic lipid molecules or other molecule inhibiting MDGI expression or activity can in some embodiments serve as the 'moiety" in the CooP conjugates described herein.
  • a difference in the amount of MDGI in the sample of the patient relative to the control sample is indicative of the presence or grade of the brain tumor, metastatic brain lesions or other tumor types.
  • the MDGI levels in blood or fractions of blood, typically serum or plasma can be used in brain tumor diagnostics and make possible to follow up the treatment efficiency and/or tumor progression.
  • Antibodies or other molecules recognizing MDGI are useful in determining the grade of brain tumor, metastatic brain lesions or other brain tumor types. They can be used as such, or in combination with conventional determination methods, such as methods based on histology, type of vasculars, p53 status, amount of proliferating cells or epidermal growth factor receptor EGFR.
  • the antibodies or other molecules that recognize MDGI are also useful in a method for determining the presence or grade of brain tumor or metastatic brain lesions or other tumor types in a patient.
  • the level of MDGI is determined in a biological sample obtained from the patient, and compared with the level of MDGI in a control sample. A difference in the amount of MDGI in the sample relative to the control sample is indicative of the presence or grade of brain tumor or other tumor types.
  • MDGI can be used in a method for determining the presence or grade of other tumor types than brain tumor.
  • Such "other tumor types” are preferably kidney carcinomas, sarcomas, ovarian carcinomas, pancreatic carcinomas, testicular cancers, melanomas and prostate carcinomas.
  • the anti-MDGI antibodies, peptides or other molecule binding MDGI can also be used in a method for diagnosing invasive brain tumor or metastatic brain lesion in a patient, or other tumor types.
  • the method includes conjugating an anti-MDGI antibody or peptide or other molecule recognizing MDGI, to a detectable label, such as an imaging agent.
  • the labeled anti-MDGI antibody or the peptide binding to MDGI is administered to the patient, and the label is detected.
  • anti-MDGI antibodies for diagnosing or treating other tumor types, for use in targeting pharmaceutically active agents to other tumor types for use in inhibiting MDGI expression or function in other tumor types.
  • the anti-MDGI antibody or other molecule recognizing MDGI can be used to target pharmaceutically active agents to brain tumor or metastatic brain lesions or other tumor types.
  • inhibitory nucleic acid molecules such as siR A or shRNA to inhibit MDGI expression or function in other tumor types.
  • An antibody or other molecule that recognizes MDGI can be used to target imaging agents or anti-cancer therapies to tumor tissue. More specifically, for example the CooP peptide or an antibody or other molecule or peptide that recognizes MDGI can be used to target imaging agents or anti-cancer therapies to tumor tissue.
  • the methods described herein encompass the use of the MDGI levels for monitoring the efficacy of therapy or relapse after the surgical removal of other tumor types than brain tumor.
  • “Determining the level of MDGI” reflects the effectiveness by which MDGI is expressed, which indicates the invasiveness or aggressiveness of the tumor. Determination of the level of MDGI can be carried out by using anti-MDGI or other molecules recognizing MDGI, for example peptides binding to MDGI.
  • anti-MDGI antibody is conjugated to a detectable label.
  • the labeled anti-MDGI antibody is administered to the patient, and the label is detected.
  • the methods for targeted therapy can in some embodiments be combined with conventional cancer therapies.
  • Such therapies are for example radiation and anti-cancer or anti-angiogenic therapies.
  • the method further comprises that the targeted therapy is combined with surgery.
  • the method the present with boron neutron capture therapy (BNCT) where tumor cells are irradiated while surrounding healthy tissues are preserved.
  • BNCT boron neutron capture therapy
  • Boron- 10 atoms are capable of capturing low- energy neutrons which leads to a nuclear reaction emitting alpha- and lithium recoil particles.
  • a high intracellular boron- 10 concentration is needed at the time of neutron irradiation to maximize a high radiation dose to the tumor.
  • the molecules of the present invention can be used as delivery molecules for boron. After infusion of the anti-MDGI antibody, other molecule or peptide, typically 1 hour after infusion, the tumor is irradiated with neutrons using optimally angled beams.
  • the disclosed methods include the determination, identification, indication, correlation, diagnosis, prognosis, etc. (which can be referred to collectively as "identifications") of subjects, diseases, conditions, states, etc. based on measurements, detections, comparisons, analyses, assays, screenings, etc.
  • identifications For example, detection of MDGI in brain tissue and/or measurement of a higher level of MDGI in brain tissue can identify a subject having MDGI-related cancer or a subject that can be treated with an anti-MDGI based treatment or MDGI-targeted treatment.
  • identifications are useful for many reasons. For example, and in particular, such identifications allow specific actions to be taken based on, and relevant to, the particular identification made.
  • diagnosis of a particular disease or condition in particular subjects has the very useful effect of identifying subjects that would benefit from treatment, actions, behaviors, etc. based on the diagnosis.
  • treatment for a particular disease or condition in subjects identified is significantly different from treatment of all subjects without making such an identification (or without regard to the identification). Subjects needing or that could benefit from the treatment will receive it and subjects that do not need or would not benefit from the treatment will not receive it.
  • methods comprising taking particular actions following and based on the disclosed identifications.
  • methods comprising creating a record of an identification (in physical— such as paper, electronic, or other— form, for example).
  • creating a record of an identification based on the disclosed methods differs physically and tangibly from merely performing a measurement, detection, comparison, analysis, assay, screen, etc.
  • Such a record is particularly substantial and significant in that it allows the identification to be fixed in a tangible form that can be, for example, communicated to others (such as those who could treat, monitor, follow-up, advise, etc.
  • identifications can be made, for example, by the same individual or entity as, by a different individual or entity than, or a combination of the same individual or entity as and a different individual or entity than, the individual or entity that made the record of the identification.
  • the disclosed methods of creating a record can be combined with any one or more other methods disclosed herein, and in particular, with any one or more steps of the disclosed methods of identification.
  • identification of a subject as having a disease or condition with a high level of a particular component or characteristic can be further identified as a subject that could or should be treated with a therapy based on or directed to the high level component or characteristic.
  • a record of such further identifications can be created (as described above, for example) and can be used in any suitable way.
  • Such further identifications can be based, for example, directly on the other
  • identifications a record of such other identifications, or a combination.
  • Such further identifications can be made, for example, by the same individual or entity as, by a different individual or entity than, or a combination of the same individual or entity as and a different individual or entity than, the individual or entity that made the other identifications.
  • the disclosed methods of making a further identification can be combined with any one or more other methods disclosed herein, and in particular, with any one or more steps of the disclosed methods of identification.
  • methods comprising treating, monitoring, following-up with, advising, etc. a subject identified in any of the disclosed methods.
  • methods comprising treating, monitoring, following-up with, advising, etc. a subject for which a record of an identification from any of the disclosed methods has been made.
  • particular treatments, monitorings, follow-ups, advice, etc. can be used based on an identification and/or based on a record of an identification.
  • a subject identified as having a disease or condition with a high level of a particular component or characteristic can be treated with a therapy based on or directed to the high level component or characteristic.
  • Such treatments, monitorings, follow-ups, advice, etc. can be based, for example, directly on identifications, a record of such identifications, or a combination.
  • Such treatments, monitorings, follow-ups, advice, etc. can be performed, for example, by the same individual or entity as, by a different individual or entity than, or a combination of the same individual or entity as and a different individual or entity than, the individual or entity that made the identifications and/or record of the identifications.
  • the disclosed methods of treating, monitoring, following-up with, advising, etc. can be combined with any one or more other methods disclosed herein, and in particular, with any one or more steps of the disclosed methods of identification.
  • MDGI was found to be the receptor/binding partner for the CooP peptide.
  • astrocytomas a class of malignant brain tumors, acquire their blood supply by co-opting existing normal blood vessels and propagating along them without initiating angiogenesis. This leads to diffuse invasion of tumor cells over long distances in the brain without formation of real tumor masses.
  • the most malignant form of astrocytomas also called glioblastoma multiforme (GBM)
  • GBM glioblastoma multiforme
  • In vivo phage display is a powerful method to isolate homing peptides and map the vascular diversity of tumors as well as other organs. Efficient tissue diffusion and good target accessibility make homing peptides excellent probes for biodistribution studies and noninvasive tumor imaging. Phage displayed peptide libraries were used to identify a peptide that very specifically homes to malignant brain tumors.
  • MDGI mammary-derived growth inhibitor
  • MDGI/H-Fabp/Fabp3 mammary-derived growth inhibitor
  • a yeast-two-hybrid screen was performed.
  • the peptide sequence was introduced into a bait plasmid and co-transformed to bacteria with the target plasmids encoding a murine embryonic (El 2.5) cDNA library.
  • Three of the hits represented two different cDNAs for the mammary-derived growth inhibitor (MDGI; NM_004102) also known as heart type fatty acid binding protein (H-FABP or FAPB-3).
  • MDGI expression in the transiently transfected U20S cells increased the CooP displaying phage binding 10-fold compared to the original library.
  • mice carrying intracranial xenografts were perfused with FITC-lectin (from Lycopercicon esculanta).
  • FITC-lectin from Lycopercicon esculanta
  • anti-MDGI antibody was administered intravenously to intracranial tumor bearing mice (U87MG) followed by histological analysis of tissues.
  • the anti-MDGI antibody accumulated in the tumor tissue but could not be detected in any other tissue studied, while the control antibody (Goat IgG) did not accumulate in tumor vasculature or in any other tissue examined.
  • Hif- la-deficient (HIFko) mouse astrocytes were propagated as previously described (Blouw et al, Cancer Cell, 4: 133-146 (2003)).
  • MDA-MB-231 breast carcinoma cells were cultured in the RPMI 1640 supplemented with 10% FCS, 2 mM L- glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • U87MG human glioma cells and BT 4 C rat glioma cells were maintained in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • U2-OS osteosarcoma cells (a kind gift from Dr. Marikki Laiho, University of Helsinki, Finland) were cultured in DMEM and 15 % FCS. All cells were cultured in a humidified 5% C0 2 atmosphere at 37°C.
  • mice Athymic female Balb/c nu-nu mice (4-6 wks) from Taconic Europe were used in all tumor experiments. Establishment of the intracranial tumors has been described (Blouw et al. , 2003). In brief, 50 000 HIFko, VEGF+, U87MG, U87-GFP, U87-MDGI- GFP or BT 4 C tumor cells in phosphate buffered saline (PBS) or DMEM were injected through the scull using a Hamilton syringe (no 84855), 2 mm right from the bregma at the depth of 3 mm in the brain parenchyma. Mice were used for some experiments 10-15 days after tumor cell inoculation.
  • PBS phosphate buffered saline
  • DMEM phosphate buffered saline
  • Mice were used for some experiments 10-15 days after tumor cell inoculation.
  • mice were used 10-21 days after tumor cell inoculation.
  • 2-5xl0 6 cells were grafted subcutaneous ly on the abdominal side of the mice (in some experiments) or into the mammary fat pad, in other experiments. All animal studies were conducted according to the guidelines of the Provincial Government of Southern Finland and the protocol was approved by the Experimental Animal Committee.
  • the bound phage was rescued and amplified in E. coli (BLT5615, Novagen, USA), and used for a second round of ex vivo selection.
  • the ex vivo enriched phage pool was injected into the tail vein of intracranial HIFko tumor- bearing mice, and allowed to circulate for 15 min.
  • animals were perfused through the left ventricle of the heart with PBS. Brain, including the tumor, was excised, and the recovered and amplified phage was used for the next found of in vivo panning.
  • the in vivo selection procedure was repeated three times. In each round, non-recombinant control phage was injected to a separate mouse to assess the background.
  • the relative phage titers were determined as the ratio of specific phage and non-recombinant control phage recovered from the tumor tissue.
  • Tumor specific peptide C00P (NH 2 -ACGLSGLGVA-CONH 2 ; SEQ ID NO:8) and its control ( H 2 -ACVAALNADG-CONH 2 ; SEQ ID NO:7) were synthesized using an Apex 396 DC multiple peptide synthesizer (Advanced ChemTech, Louisville, KY) with Fmoc strategy and 0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro- phosphate (HBTU, GLS Biochem, Shanghai, China) and N,N-diisopropylethylamine (DIPEA, Fluka, Steinheim, Germany) as coupling reagents.
  • Apex 396 DC multiple peptide synthesizer Advanced ChemTech, Louisville, KY
  • DIPEA N,N-diisopropylethylamine
  • Rink resin was used as the solid phase (Novabiochem, Laufelfingen, Switzerland) to produce carboxyl terminal amide. Synthesized peptides were left on resin for further conjugations.
  • DTPA was conjugated directly to the alpha-amino group of the peptide using tertbutyl protected DTPA (diethylenetriamine-N,N,N",N"-tetra-/er/-butyl acetate-N'-acetic acid,
  • Isothiocyanate activated fluorescein (FITC; Sigma, St. Louis, MO) was conjugated to the peptides on resin via the side chain of the additional amino terminal lysine to avoid the spontaneous cleavage during the strong acid treatment.
  • FITC fluorescein
  • DMF dimethyl formamide
  • Mtt acid labile methyltrityl
  • FITC (2 mg from the stock solution of 10 mg/ml in DMF) was added into reaction mixture following addition of 15 ⁇ triethanolamine (TEA). Resin was mixed for 1 h. FITC conjugation step was repeated to gain maximal yield of FITC conjugated peptides. Conjugated peptides were cleaved from the resin using a mixture of 95 % TFA, 3 % ethanedithiol (EDT), 1 % triisopropylsilane (TIS) and 1 % H 2 0 where EDT and TIS were used as scavengers.
  • EDT ethanedithiol
  • TIS triisopropylsilane
  • Peptides were purified with RP-HPLC (Shimadzu, Kioto, Japan) using C 18 reverse phase column (xTERRA, 250 x 10 mm, Waters, Milford, MA), and acetonitrile gradient (ACN, 0-95 %, 45 min).
  • the peptides were verified by mass spectrometry on an ABI QSTAR XL hybrid mass spectrometer using MALDI interface (Applied Biosystems, Foster City, CA) and the purity was determined by an analytical HPLC
  • the DTPA conjugated peptides (20 ⁇ g per animal) were mixed with 0.2 M NaAc (pH 5) and 5 MBq of i n Indiumchloride ( m InCl 3 , Mallinckrodt, Le Petten, Netherlands) per animal was added to the mixture. The total volume of the solution was calculated to be 100 ⁇ /animal.
  • the reaction mixture was incubated for an hour at RT and the radiochemical purity was measured by using an instant thin-layer chromatography (ITLC-SG, Pall Corporation, USA) on a 13 x 1.5 cm strip using 0.3 M NaCit (pH 5) as a mobile phase.
  • the radiochemical purity of the peptides was 99 - 100 %.
  • SPECT/CT single photon emission computed tomography-computed tomography
  • imaging animals were anesthetized with 1-3% isoflurane in N2/O2 at a 70:30 ratio.
  • the radiolabeled peptide (100 ⁇ ) was injected into the tail vein followed by SPECT imaging using a combined small animal SPECT/CT (Gamma Medica Inc., Northridge, CA) with dual gamma camera modality.
  • Medium energy parallel hole collimators were used in all gamma imaging with a 171/245/416 keV energy window for m In. Planar two-dimensional imaging for all animals was performed at earliest possible time point after injection with 60 s acquisition time/image up to 30 minutes.
  • planar SPECT images were combined to X-ray image with an in-house Matlab software (The MathWorks Inc. Natick, MA).
  • Three-dimensional SPECT reconstruction was carried out using the LumGem software (Gamma Medica,
  • organs and tissues blood, heart, lungs, liver, spleen, left kidney, duodenum, ovaries, fat, muscle, skin, brain and brain tumor
  • the counts obtained were corrected for the background radiation and physical decay, and tissue radioactivity was expressed as percentage of the injected dose per gram of tissue (% ID/g) ⁇ SD.
  • Intravenously injected fluorescein-labeled peptides 100 ⁇ of 1 mM solution
  • antibodies (20 ⁇ g/animal) were allowed to circulate for one hour followed by perfusion through the heart with 10 ml of phosphate buffered saline (PBS) and 10 ml of 4% paraformaldehyde (PFA) in PBS.
  • PBS phosphate buffered saline
  • PFA paraformaldehyde
  • Tumors and organs were excised, PFA-fixed and soaked in 30% sucrose (w/v) overnight.
  • Tissues were frozen in the OCT embedding medium (Tissue-Tek). Sections (5-10 ⁇ ) were cut for histological analyses.
  • GATCCAGCAACGCCTAACCCGCTCAGTCCGCAG (SEQ ID NO:2)) purchased from Sigma encoding the CooP sequence were cloned into the pGBKT7 bait vector (Clontech).
  • a mouse embryonic (E12.5) library in the Pad-GAL4-2.1 prey vector was transformed into yeast cells expressing the baits. Clones producing ⁇ -galactosidase were isolated, and the prey cDNAs were purified and sequenced. The screen was performed according to the Fields' method (Fields and Song, Nature, 340:245-246 (1989)) at the Yeast Two-Hybrid Core Facility, Biomedicum Helsinki (University of Helsinki, Finland).
  • Full- length MGDI was cloned using the tumor-derived cDNA as a template with the following primers: fw: GGAATTCGCGGACGCCTTTGTCGGTACCTGGAAG (SEQ ID NO:3); rev: CCTCGAGTCACGCCTCCTTCTCATAAGTCCGAGTGCTC (SEQ ID NO:4), and ligated to the pcDNA3-9E10 plasmid (a kind gift from Dr. Paivi M. Ojala, University of Helsinki, Finland).
  • U2-OS osteosarcoma cells were transfected with the pcDNA3-9E10 plasmid encoding the full-length MDGI with the FuGENE transfection reagent (Roche) according to the manufacturer's instructions.
  • Cells were plated on 10 cm 0 cell culture dishes 24 hours prior to transfection.
  • Phage displaying either the CooP peptide sequence CGLSGLGVA (SEQ ID NO: 5) or random CX 7 C library sequences were incubated with the transfected cells for two hours at 4°C.
  • the unbound phage was removed with extensive washes in PBS/1% BSA. Cells were detached using a plastic scraper and the bound phage was rescued by infecting with bacteria.
  • the binding affinities were determined by phage titration as the ratio of CooP or library phage and non-recombinant control phage recovered from the cells.
  • MDA-MB-231 breast tumor cell line stably expressing the MDGI as a c-Myc fusion protein cells were transfected with the pcDNA3-9E10-MDGI plasmid as described herein. 24-48 h post-transfection cells were exposed to the G418 (0.7 mg/ml, Sigma) selection. Geneticin-resistant clones were picked by the ring-cloning method, expanded, and subsequently tested for the presence of the transgene expression by immunofluorescence and by Western-blot analyses.
  • MDGI gene was cloned into a lentiviral pBOB ⁇ cag ⁇ GFP expression vector (kind gift from Dr. Yla-Herttuala, University of Eastern Finland, Kuopio, Finland) and transfected into human embryonic kidney cells (293FT, 4 x 10 6 ) using the
  • LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA). Virus containing supernatants were collected 72 hours post-transfection. To remove cell debris the medium was centrifuged briefly (2 min, 160 g) and filtered through a 0.45 ⁇ filter. The supernatant was concentrated with the Optima L-80 XP ultracentrifuge (Beckmann) supplied with a swinging bucket rotor (SW28, Beckmann coulter). MDA-M-231 and U87MG cells (50% confluent) were transduced with concentrated MDGI-GFP and GFP encoding viruses.
  • Moderately and highly transgene positive cells were sorted by using a BD FACSAria cell sorter (purity mode, BD Biosciences, USA). Expression of the trans genes was verified using Western blot-analyses. The efficiency of transduction was determined using immunofluorescence microscopy by counting the percentage of the transgene expressing cells.
  • the following primary antibodies were used: goat or rabbit polyclonal anti- MDGI antibody (1 :200, Santa Cruz Biotechnology, USA), mouse monoclonal anti-c- Myc (9E10) antibody (1 : 1000, Biocompare), rabbit polyclonal anti-GAPDH antibody (1 : 1000, Cell Signaling, USA). After extensive washes the membranes were incubated with the horseradish peroxidase-coupled anti-goat, anti-mouse or anti-rabbit antibodies (Dako, Denmark). The bound antibody was detected with the ECL (Pierce). Immunohistochemistry of paraffin-embedded tissues Five-micrometer thick, paraffin-embedded tissue sections were prepared for histological analysis.
  • Antigen retrieval was performed by heating the samples in a 10 mM sodium citrate buffer (pH 6.0) using the microwave oven (780W 5 min, 380W 10 min). MDGI was detected from the sections using the TSA Indirect Kit (PerkinElmer, Boston, MA, USA). Nuclei were stained with Hematoxylin.
  • Formalin- fixed paraffin-embedded tumor tissue samples obtained from patients diagnosed with a brain tumor following craniotomy were retrieved from the archives of the Department of Pathology, Helsinki University Central Hospital, Finland.
  • the brain tumors were originally diagnosed as either pilocytic astrocytoma (grade 1), diffuse astrocytoma (grade 2), anaplastic astrocytoma (grade 3), primary or secondary glioblastoma multiforme GBM (grade 4), medulloblastoma or ependymoma.
  • the histopathological diagnoses were reviewed by professional pathologists (AP and OT) based on the World Health Organization (WHO) criteria.
  • the study was approved by an Ethics Committee of the Helsinki University Central Hospital, Helsinki Uusimaa, Finland. A permission to use tumor tissue for the study was obtained from the Ministry of Social Affairs and Health, Finland.
  • Type I collagen from rat tail (C7661, Sigma) was dissolved in 0.2% acetic acid. The collagen was diluted to a final concentration of 2.2 mg/ml in MEM and neutralized with NaOH. Collagen gels were then casted into the upper chamber of cell culture inserts (Falcon) and allowed to polymerize at +37°C for 1 hour. MDA-MB-231 breast carcinoma cells (1.2xl0 5 ) in complete RPMI 1640 medium supplemented with 10% FBS were laid on top of the gels. Complete RPMI 1640 medium was added to the lower chamber and the cells were cultured at 37°C for 14 days, replenishing the medium every second/third day. The gels were then fixed with 3% PFA and embedded into TissueTek.
  • FITC-CooP 100 ⁇ , 100 ⁇ /animal was injected into the tail vein of i.c HIFko astrocytoma-bearing mice and allowed it to circulate for 60 minutes.
  • the peptide accumulated in the tumor islets in the brain (data not shown).
  • Adjacent tissue sections were stained for the presence of the SV40 large T antigen to visualize the tumor cells (data not shown).
  • the CooP peptide was not detected in the surrounding histologically normal brain or other tissues examined (data not shown).
  • CooP peptide To assess tumor-type specificity of the CooP peptide, its homing to other types of brain tumors was tested.
  • the CooP peptide homed efficiently to the intracranial U87MG human glioma xenografts (data not shown), and to a lesser yet detectable extent to the rat BT 4 C glioma xenografts (data not shown) after an intravenous injection.
  • No CooP peptide was detected in the orthotopic MDA-MB-231 human breast cancer xenografts (Figure 2F).
  • the CooP peptide did not home to tumors established from the HIFko cells when they were implanted subcutaneous ly (data not shown).
  • CooP peptide did not home to astrocytomas arising from same cells than HIFko tumors but overexpressing VEGF that contain massive network of angiogenic blood vessels (data not shown).
  • Mammary-derived growth inhibitor is the putative receptor for the CooP peptide. Due to its very specific homing the CooP peptide should bind to a receptor(s)/binding partner(s) on the target tissue.
  • a yeast-two-hybrid screen was used (Fields and Song, 1989).
  • the peptide sequence was introduced into the bait plasmid and co-transformed to bacteria with the target plasmids encoding a murine embryonic (E12.5) cDNA library.
  • An embryonic library was selected since the CooP peptide did not home to the normal brain and many embryonic proteins become upregulated in tumor tissues but are not present in the normal adult tissues (e.g.
  • c-Myc the EDB domain of oncofetal fibronectin (Carnemolla et al, J Cell Biol 108, 1 139-1148 (1989), Castellani et al, Int J Cancer 59, 612-618 (1994), Kaczmarek et al, Int J Cancer 59, 11-16 (1994), Zardi et al, EMBO J 6, 2337-2342 (1987)) and the large isoform of tenascin C (Borsi et al, Int J Cancer 52, 688-692 (1992),Carnemolla et al, Eur J Biochem 205, 561-567 (1992)).
  • the yeast-two-hybrid screen yielded ten in-frame hits, which were used for sequence identification from the BLAST database (internet site
  • cDNAs represented four different proteins: (i) a eukaryotic translational initiation factor (Eif3s7), (ii) a neural peptidase (Cpe), (iii) a mammary-derived growth inhibitor (MDGI) and (iv) an EST.
  • Eif3s7 a eukaryotic translational initiation factor
  • Cpe neural peptidase
  • MDGI mammary-derived growth inhibitor
  • EST an EST.
  • Three of the hits represented two different cDNAs for the MDGI.
  • MDGI expression in the transiently transfected U20S cells increased the binding of CooP displaying phage 10-fold compared to the original library (FIG. 2A).
  • MDGI expression in transfected cells was confirmed by Western blot analyses (FIG. 2B).
  • Fabp3 fatty-acid binding protein 3
  • H-FABP heart fatty acid-binding protein
  • MDGI should be expressed either by the tumor cells and/or tumor-associated stromal cells.
  • FITC-tagged CooP peptide was injected i.v. into the intracranial HIFko and U87MG tumor-bearing mice. Tumor sections were then studied for the presence of the peptide as well as for the MDGI expression using an antibody against MDGI.
  • the MDGI protein was detected both in the U87MG and HIFko brain tumors. Furthermore, partial co- localization of the MGDI and the peptide was observed in the tumor tissue (data not shown).
  • the homing pattern of the CooP peptide after systemic delivery varied in different i.e. tumors; in the HIFko tumor islets the peptide showed diffuse distribution throughout the islet (data not shown) while in the U87MG human glioma xenografts the peptide displayed a vessel-like homing pattern (data not shown). Importantly, MDGI was expressed in all tumors the CooP peptide homed to (i.e.
  • HIFko, U87MG, and BT 4 C tumors were found in the tumors to which the CooP peptide did not home (i.c.VEGF-overexpressing tumor, orthotopic MDA-MD-231 breast cancer xenograft and subcutaneous HIFko tumor; data not shown).
  • an MDGI expressing MDA-MB-231 tumor cell line was established and used to monitor whether the presence of MDGI would allow the CooP homing to the MDA-MB-231 tumors.
  • the CooP peptide homed with high efficiency to all MDGI-expressing xenografts analyzed (data not shown) while only negligible amount of the peptide was detected in the parental MDA-MB-231 tumor (data not shown).
  • the presence of MDGI in the tumor tissue increased substantially the homing of the CooP peptide to these tumors.
  • MDGI being the cardiac isoform of the fatty-acid binding proteins is abundantly expressed in the muscle and heart, and to a lesser extent, in the brain (Zschiesche et ah, Histochem Cell Biol, 103: 147-156 (1995)). Therefore the MDGI expression in the U87MG brain tumor-bearing mice was studied. To visualize the blood vasculature these mice were perfused with FITC-lectin (from Lycopercicon esculanta). MDGI was expressed in the tumor (data not shown), muscle (data not shown) and heart (data not shown) but MDGI expression was not detected in the brain with the antibody and staining method used (data not shown). Interestingly, MDGI showed vasculature- associated expression only in the tumors (data not shown).
  • Circulating anti-MDGI antibody localizes to tumor-associated blood vessels
  • the CooP peptide selectively homed to a subtype of brain tumors when expressed as a part of the phage capsid protein or when conjugated to a fluorescein label. This prompted us to investigate whether antibodies against MDGI, the potential binding partner of the peptide, would also accumulate in tumors. Furthermore, MDGI should be accessible via the circulation to act as a receptor for the systemically administered peptide.
  • the anti-MDGI antibody was administered intravenously to the i.e. U87MG tumor-bearing mice.
  • DTPA conjugated CooP-peptide was labeled with m In and injected it (20 ⁇ g in 100 ⁇ ) intravenously into i.c.U87MG tumor-bearing mice. 2D images were collected from all animals up to 15-30 min post-injection. A 3D image was reconstructed 60 min, 120 min or 24 hrs post-injections. The plasma clearance of the peptide was rapid since most of the activity was excreted in the urine within the first 20 minutes (data not shown).
  • Tumor site radioactivity was approximately 20-fold higher at 15 min post- injection and about 12-fold higher at two hours post-injection compared to the contralateral brain hemisphere, indicating marked CooP peptide accumulation in the malignant tissue (FIG. 3 A). Importantly, only brain tumors showed increased accumulation of the CooP peptide compared to the control peptide at 2 hrs post-injection (Figure 3B). Tumor site radioactivity was increased over healthy brain at all times measured (Figure 3B).
  • MDGI expression in human brain tumors has not been reported earlier.
  • the presence and localization of MDGI in clinical brain tumor samples was investigated by immunohistochemistry.
  • a small panel of astrocyte-derived tumor samples from patients who had undergone craniotomy was analyzed (Table 1).
  • Normal brain tissue (data not shown) and pilocytic astrocytomas (grade 1) did not express MDGI at detectable levels (data not shown).
  • the majority of the grade 2 astrocytomas (60%) (data not shown) and 80% of anaplastic astrocytomas (data not shown) showed moderate levels of MDGI expression mostly in the vasculature and in the perivascular compartment.
  • glioblastomas 17/18 (94%) expressed very high levels of MDGI.
  • the expression was concentrated on perinecrotic areas of the tumors (data not shown), i.e., all but one of the glioblastomas expressed very high levels of MDGL
  • the expression was concentrated on perivascular and perinecrotic areas of the tumors (data not shown).
  • Vasculature-associated MDGI was also found in some glomeruloid- and slit-like vessels that are typical for the GBM (data not shown). Sixty- seven percent of ependymomas were moderately positive while medulloblastomas () showed no MDGI expression (data not shown).
  • MDGI expression in human brain tumors is presented in Table 1.
  • the levels of MDGI in the serum of MDGI-expressing orthotopic glioma and subcutaneous MDA-MB-231 breast carcinoma xenograft-bearing mice were compared to that of healthy animals using Western blot analyses.
  • the MDGI-expressing xenograft bearing mice had considerable amounts of MDGI in the serum, whereas no MDGI was detected in the serum of healthy animals. This indicates that the serum or plasma levels of MDGI are useful as surrogate marker for brain tumor diagnosis and the efficacy of the therapy.
  • Prominin (CD 133) positive cells were isolated from the commercially available human glioma cell line U87MG and created a CD133-positive cell line. These cells should represent the glioma stem or initiating cells.
  • the human breast cancer xenografts derived from the MDA-MB-231 cells expressed MDGI at very low levels; therefore stable MDA-MB-231 cell lines expressing moderate or high levels of the MDGI protein were created. Xenograft tumors of these stable MDGI-MDA cells were established and their growth rate and metastatic potential compared to that of MDA-MB-231 xenografts. The data showed that tumors arising from cells expressing moderate levels of MDGI grew similarly to the MDA-MB-231 tumors while tumors generated from the high MDGI expressing cells grew well, initially, but then started to regress.
  • the data from these studies show that MDGI expressing brain tumors and breast carcinomas show increased invasion and metastatic capacity compared to the parental tumors in vivo.
  • the data also showed increased invasive potential of MDGI expressing breast carcinoma cells in vitro. Accordingly, tumor invasion and metastasis can be reduced by reducing the MDGI expression.
  • This can be performed for example by using the R Ai technology or possibly using antibodies against MDGI.
  • ShR A or SiRNA being capable of specifically silencing MDGI expression, can be produced by methods well known to a person skilled in the art. Also antibodies recognizing MDGI can be used to reduce tumor invasion and metastasis.
  • Targeted treatment prolongs the survival of the brain tumor bearing mice.
  • Tumor bearing immunocompromised mice treated with saline (PBS), free chlorambusil (Cbl) or targeted drug showed marked differences in response to treatment as measured by the weight gain from the normalized weight at tumor cell implantation.
  • PBS saline
  • Cbl free chlorambusil
  • targeted drug chlorambusil conjugated to CooP peptide; CooP-CPP-Cbl, 5 mg/kg
  • MDGI mammary-derived growth inhibitor
  • H-FABP the heart isoform of the fatty acid-binding proteins
  • MDGI expression was analogous with the homing of CooP. MDGI was expressed only in tumors the CooP peptide homed to. Accordingly, tumors the CooP peptide did not home to were negative for MDGI expression.
  • the expression of MDGI and the homing of the peptide seemed to be dependent on the tumor microenvironment since the HIFko tumors in the subcutaneous space did not express the receptor and the peptide did not home to those tumors in contrast to the same tumors grown in the intracranial space. This is in line with the pre-existing data showing that the tumor microenvironment affects maturation of the vasculature and that the molecular composition of blood vessels at diverse locations is different (Madri et al, 1983, Janzer et al, 1987, Aird et al, 1997, Blouw et al. 2003).
  • the binding of the Coop peptide to the MDGI transfected cells compared to the non-selected peptide library was markedly higher in vitro, and MDGI overexpression dramatically increased the homing of the peptide to the tumor xenografts in vivo.
  • circulating antibodies against the MDGI accumulated only in the tumor tissue but were non-detectable in other tissues indicating MDGI to be adequately accessible through the circulation and exposed to intravenously injected ligands.
  • MDGI was also expressed in heart and skeletal muscle as previously described (Zschiesche et al, Histochem Cell Biol, 103 : 147-156 (1995)).
  • Low levels of MDGI were observed in the lining epithelia of brain ventricles and choroid plexus but importantly, vascular expression was restricted to the tumor tissue allowing the tumor specific homing of the peptide.
  • tissue-specific vascular expression is the membrane dipeptidase (MDP), the receptor for the lung-homing peptide GFE-1.
  • MDP membrane dipeptidase
  • MDP is also expressed in the proximal tubules of the kidney but there it is not expressed in the vascular compartment and therefore inaccessible via vascular targeting (Rajotte et al, Journal of Clinical Investigation, 102:430-437 (1998); Rajotte and Ruoslahti, Journal of Biological Chemistry, 21 A: 11593-1 1598 (1999)).
  • the FABPs are primarily intracellular proteins, although membrane-associated forms have also been reported (Glatz and van der Vusse, rog Lipid Res, 35:243-282 (1996)) that mediate the fatty acid and/or hydrophobic ligand uptake, transport and targeting in their respective tissues (Veerkamp, Proc Nutr Soc, 54:23-37 (1995)). In cells transiently overexpressing the MDGI, a small fraction of the protein was detected on the surface although the majority of the protein localized to the cytoplasm.
  • MDGI appears to be the only FABP that affects cell proliferation and differentiation. This function might be separate from its ligand binding function since it can be mimicked by a C-terminal peptide of MGDI, which cannot bind fatty acids (Yang et al, J. Cell Biol. 127: 1097-1 109 (1995), Wang and Kurtz, Oncogene 19(20):2455-60 (2000), Storch and Corsico, Annu. Rev. Nutr. 28:73-95 (2008)).
  • the region of the genome coding for the MDGI is often deleted in sporadic breast cancers and MDGI appears to be
  • MDGI inhibits cell growth in culture and reduces the tumorigenicity of the MCF-7 breast cancer cells (Huynh and Pollak, In Vitro Cell Dev Biol Anim., 31 :25-29 (1995)).
  • MDA-MB- 231 breast cancer cells MDGI expression appears to inhibit invasion and adhesion (Nevo et al. Oncogene 29(49):6452-63 (2010)).
  • Tumor half to healthy brain half ratio was up to 20: 1 indicating outstanding accumulation of the peptide in the tumor tissue while the whole- body radioactivity remained minimal.
  • peptide-based imaging strategies have been "dominated” by the RGD-peptide or its derivatives targeting the ⁇ 3 integrins in the angiogenic vessels, and the somatostatin receptor specific peptide analogues "hitting" the neuroendocrine tumors (reviewed in Reubi and Maecke, 2008).
  • a subset of tumors including low-grade astrocytomas, initially grows by co- opting existing blood vessels. Glioblastomas first regress the co-opted host vasculature before they initiate the growth of new blood vessels i.e. angiogenesis.
  • Angiopoietin 2 (Ang-2) is upregulated in the co-opted blood vessels prior their regression and it is associated with the vascular regression in the absence of vascular endothelial growth factor (VEGF) expression (Holash et al, Science, 284: 1994-1998 (1999), Holash et al, Oncogene, 18:5356-5362 (1999)).
  • VEGF vascular endothelial growth factor
  • Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des méthodes et des compositions destinées à évaluer des tumeurs cérébrales ou des lésions cérébrales métastatiques ou d'autres types de tumeur cérébrale chez un patient en détectant la présence, le siège, et/ou les taux de MDGI chez le patient ou dans des échantillons provenant du patient. Par exemple, la présence ou le degré de la tumeur cérébrale ou des lésions cérébrales métastatiques ou d'autres types de tumeur cérébrale chez un patient peuvent être déterminés. La présente invention concerne également des méthodes et des compositions destinées à traiter des patients atteints de tumeur cérébrale ou de lésions cérébrales métastatiques ou d'autres types de tumeur cérébrale par ciblage thérapeutique du site de la tumeur cérébrale ou des lésions cérébrales métastatiques ou d'autres types de tumeur cérébrale. La présente invention concerne également des méthodes et des compositions destinées à surveiller l'efficacité de la thérapie ou la rechute après l'ablation chirurgicale de la tumeur cérébrale ou de la lésion métastatique ou d'autre type de tissu tumoral dans le cerveau. Ces méthodes peuvent utiliser un anticorps anti-MDGI et/ou un peptide CooP pour cibler le MDGI dans la tumeur cérébrale ou les lésions cérébrales métastatiques ou d'autres types de tumeur cérébrale. Le taux de MDGI détecté dans un échantillon comparativement au taux de MDGI dans un échantillon de contrôle indique la présence ou le degré de la tumeur cérébrale ou d'autre type de tumeur cérébrale.
PCT/US2013/038219 2012-04-25 2013-04-25 Diagnostic et traitement de tumeur cérébrale WO2013163431A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/396,414 US9581598B2 (en) 2012-04-25 2013-04-25 Diagnosis and treatment of brain tumor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261638064P 2012-04-25 2012-04-25
US61/638,064 2012-04-25

Publications (1)

Publication Number Publication Date
WO2013163431A1 true WO2013163431A1 (fr) 2013-10-31

Family

ID=49483892

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/038219 WO2013163431A1 (fr) 2012-04-25 2013-04-25 Diagnostic et traitement de tumeur cérébrale

Country Status (2)

Country Link
US (1) US9581598B2 (fr)
WO (1) WO2013163431A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016193757A1 (fr) * 2015-06-03 2016-12-08 Oxford University Innovation Limited Procédé pour diagnostiquer une tumeur cérébrale chez un être humain
US10401363B2 (en) 2014-12-19 2019-09-03 Althia Health, S.L. Monoclonal antibody for the diagnosis, treatment and/or prevention of brain tumors and brain lesions

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020127619A1 (en) * 1998-12-01 2002-09-12 Marti Jett Method of diagnosing stage or aggressiveness of breast and prostate cancer based on levels of fatty acid binding proteins
EP1669451A1 (fr) * 2003-09-01 2006-06-14 Japan Science and Technology Agency Marqueur de tumeur cerebrale et procede pour diagnostiquer une tumeur cerebrale

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020127619A1 (en) * 1998-12-01 2002-09-12 Marti Jett Method of diagnosing stage or aggressiveness of breast and prostate cancer based on levels of fatty acid binding proteins
EP1669451A1 (fr) * 2003-09-01 2006-06-14 Japan Science and Technology Agency Marqueur de tumeur cerebrale et procede pour diagnostiquer une tumeur cerebrale

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JONNA NEVO., NOVEL PLAYERS IN THE INTEGRIN SIGNALING ORCHESTRA: TCPTP AND MDGI., 2010, TURKU, pages 46 - 52 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10401363B2 (en) 2014-12-19 2019-09-03 Althia Health, S.L. Monoclonal antibody for the diagnosis, treatment and/or prevention of brain tumors and brain lesions
WO2016193757A1 (fr) * 2015-06-03 2016-12-08 Oxford University Innovation Limited Procédé pour diagnostiquer une tumeur cérébrale chez un être humain
US10859576B2 (en) 2015-06-03 2020-12-08 Oxford University Innovation Limited Method for diagnosing a brain tumour in a human

Also Published As

Publication number Publication date
US20150212086A1 (en) 2015-07-30
US9581598B2 (en) 2017-02-28

Similar Documents

Publication Publication Date Title
US10124077B2 (en) Plectin-1 targeted agents for detection and treatment of pancreatic ductal adenocarcinoma
Hyvönen et al. Novel target for peptide-based imaging and treatment of brain tumors
CA2779730C (fr) Compositions et procedes de detection la plectine-1 comme biomarqueur pour le cancer
Peng et al. Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells
JP2021507925A (ja) Ｇｐｃｒヘテロマー阻害剤及びその用途
JP2014510758A (ja) ほ乳類において脂肪細胞を標的とするための方法および組成物
Askoxylakis et al. Peptide-based targeting of the platelet-derived growth factor receptor beta
Khan et al. Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice
CN107249636B (zh) 以ckap4作为靶分子的抗肿瘤剂
KR101947529B1 (ko) Rhamm 결합 펩타이드
US9581598B2 (en) Diagnosis and treatment of brain tumor
WO2023179789A1 (fr) Préparation et application anti-tumorale d'un vecteur de thérapie génique interférant avec l'expression de la protéine 6 (cmtm6) à domaine transmembranaire marvel de type cklf
US11192920B2 (en) Peptides for binding to CD44v6 and use thereof
WO2017155277A1 (fr) Procédé de criblage d'un agent anticancéreux inhibant la liaison d'aimp2-dx2 et de hsp70
Schaffner-Reckinger et al. The actin-bundling protein L-plastin—A double-edged sword: Beneficial for the immune response, maleficent in cancer
TW202003013A (zh) 阻斷緊迫導致腫瘤生成之方法
WO2018008942A1 (fr) Peptide destiné à cibler le cancer du sein et utilisation associée
WO2019175376A1 (fr) Micropeptides et leurs utilisations
JPWO2005061704A1 (ja) 癌の予防・治療剤
WO2012133994A1 (fr) Procédé de dépistage destiné à un agent de traitement du cancer utilisant une interaction entre le pauf et l'un de ses partenaires de liaison
CN113195704A (zh) 用于治疗癌症的肽治疗剂及其用途
EP2578227B1 (fr) Méthode pour le traitement du cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13782316

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 14396414

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13782316

Country of ref document: EP

Kind code of ref document: A1